A DNA segment encoding the anticodon stem/loop of tRNA determines the specific recombination of integrative-conjugative elements in Acidithiobacillus species.

Horizontal gene transfer is crucial for the adaptation of microorganisms to environmental cues. The acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans encodes an integrative-conjugative genetic element (ICEAfe1) inserted in the gene encoding a tRNAAla. This genetic element is actively excised from the chromosome upon induction of DNA damage. A similar genetic element (ICEAcaTY.2) is also found in an equivalent position in the genome of Acidithiobacillus caldus. The local genomic context of both mobile genetic elements is highly syntenous and the cognate integrases are well conserved. By means of site directed mutagenesis, target site deletions and in vivo integrations assays in the heterologous model Escherichia coli, we assessed the target sequence requirements for site-specific recombination to be catalyzed by these integrases. We determined that each enzyme recognizes a specific small DNA segment encoding the anticodon stem/loop of the tRNA as target site and that specific positions in these regions are well conserved in the target attB sites of orthologous integrases. Also, we demonstrate that the local genetic context of the target sequence is not relevant for the integration to take place. These findings shed new light on the mechanism of site-specific integration of integrative-conjugative elements in members of Acidithiobacillus genus.

Resolution of Mismatched Overlap Holliday Junction Intermediates by the Tyrosine Recombinase IntDOT.

CTnDOT is an integrated conjugative element found in Bacteroides species. CTnDOT contains and transfers antibiotic resistance genes. The element integrates into and excises from the host chromosome via a Holliday junction (HJ) intermediate as part of a site-specific recombination mechanism. The CTnDOT integrase, IntDOT, is a tyrosine recombinase with core-binding, catalytic, and amino-terminal (N) domains. Unlike well-studied tyrosine recombinases, such as lambda integrase (Int), IntDOT is able to resolve Holliday junctions containing heterology (mismatched bases) between the sites of strand exchange. All known natural isolates of CTnDOT contain mismatches in the overlap region between the sites of strand exchange. Previous work showed that IntDOT was unable to resolve synthetic Holliday junctions containing mismatched bases to products in the absence of the arm-type sites and a DNA-bending protein. We constructed synthetic HJs with the arm-type sites and tested them with the Bacteroides host factor (BHFa). We found that the addition of BHFa stimulated resolution of HJ intermediates with mismatched overlap regions to products. In addition, the L1 site is required for directionality of the reaction, particularly when the HJ contains mismatches. BHFa is required for product formation when the overlap region contains mismatches, and it stimulates resolution to products when the overlap region is identical. Without this DNA bending, the N domain of IntDOT is likely unable to bind the L1 arm-type site. These findings suggest that BHFa bends DNA into the necessary conformation for the higher-order complexes, including the L1 site, that are required for product formation.IMPORTANCE CTnDOT is a mobile element that carries antibiotic resistance genes and moves by site-selective recombination and subsequent conjugation. The recombination reaction is catalyzed by an integrase IntDOT that is a member of the tyrosine recombinase family. The reaction proceeds through ordered strand exchanges that generate a Holliday junction (HJ) intermediate. Unlike other tyrosine recombinases, IntDOT can resolve HJs containing mismatched bases in the overlap region in vivo, as is the case under natural conditions. However, HJ intermediates including only IntDOT core-type sites cannot be resolved to products if the HJ intermediate contains mismatched bases. We added arm-type sites in cis and in trans to the HJ intermediates and the protein BHFa to study the requirements for higher-order nucleoprotein complexes.

The Bacteroides thetaiotaomicron protein Bacteroides host factor A participates in integration of the integrative conjugative element CTnDOT into the chromosome.

UNLABELLED: CTnDOT is a conjugative transposon found in Bacteroides species. It encodes multiple antibiotic resistances and is stimulated to transfer by exposure to tetracycline. CTnDOT integration into the host chromosome requires IntDOT and a previously unknown host factor. We have identified a protein, designated BHFa (Bacteroides host factor A), that participates in integrative recombination. BHFa is the first host factor identified for a site-specific recombination reaction in the CTnDOT family of integrative and conjugative elements. Based on the amino acid sequence of BHFa, the ability to bind specifically to 4 sites in the attDOT DNA, and its activity in the integration reaction, BHFa is a member of the IHF/HU family of nucleoid-associated proteins. Other DNA bending proteins that bind DNA nonspecifically can substitute for BHFa in the integration reaction. IMPORTANCE: Bacteroides species are normal members of the human colonic microbiota. These species can harbor and spread self-transmissible genetic elements (integrative conjugative elements [ICEs]) that contain antibiotic resistance genes. This work describes the role of a protein, BHFa, and its importance in the integration reaction required for the element CTnDOT to persist in Bacteroides host cells.